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PubMed1981

The effect of limited proteolysis on rabbit muscle creatine kinase.

Creatine kinase from rabbit muscle is inactivated by limited proteolysis with proteinase K from Tritirachium album. Gel-filtration and cross-linking studies showed that the limited proteolysis did not affect the molecular weight of the enzyme under non-denaturing conditions, but did cause changes in the reactivity of the reactive thiol group on each subunit and in the ability of the enzyme to form a 'transition-state analogue' complex in the presence of magnesium acetate plus ADP plus creatinine plus NaNO3.

Adenosine Diphosphate↗
PubMed1977

Some properties of human skeletal muscle creatine kinase.

Creatine kinase has been purified from human skeletal muscle. The properties of the human enzyme are similar to those of the enzyme from rabbit muscle. The molecular weight was determined as approximately 80000 with a probable two reactive sulphydryl groups per molecule. Manganous (II) ion was almost as effective as magnesium as the activating metal ion, and calcium and cobalt could also act in this capacity. Under standardized conditions the nucleotide specificity was ADP greater than dADP greater than IDP greater than GDP greater than UDP greater than XDP in the reverse reaction. No hydrolytic activity was observed with ATP. Initial velocity and product inhibition studies were used to determine various kinetic constants for the substrates of the enzyme. It was concluded that, as for the rabbit muscle enzyme, the reaction probably followed a rapid equilibrium random mechanism. Anomalous kinetic behaviour, however, was observed for the forward reaction for MgATP2- but not for creatine, when measurements were extended over a much wider range than normally used. The reciprocal plot of velocity as a function of substrate concentration gave a curve, concave downwards, instead of a straight line.

Creatine Kinase↗
PubMed1999

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.

Adenosine Triphosphate↗
PubMed1983

Detection of carriers of X-linked gene for Duchenne muscular dystrophy by levels of creatine kinase and pyruvate kinase.

Creatine kinase (CK) is the enzyme most often utilized for the detection of carriers of the gene for X-linked muscular dystrophies. In 1974, pyruvate kinase (PK) levels were also found to be increased in these carriers. The objective of the present study, carried out on 77 women related to patients with Duchenne muscular dystrophy (DMD), was to compare the efficiency of the two enzymes in the detection process. Of the 11 obligate heterozygotes for the DMD gene in the group, 8 exhibited elevated mean CK levels, 6 had elevated mean PK levels, and 9 had elevated mean levels of at least one of the enzymes. Among the mothers of isolated patients, 2/13 had elevated mean CK levels, 3/13 had elevated mean PK levels, and 5/13 had elevated mean levels of at least one of these enzymes. Thus, the study confirms data obtained by other investigators indicating that the use of PK can increase the detection rate of carriers of the gene for X-linked muscular dystrophies.

Clinical Enzyme Tests↗
PubMed1996

Structure of mitochondrial creatine kinase.

Creatine kinase (CK, EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the cytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 angstrom with a channel 20 angstrom wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small alpha-helical domain and a large domain containing an eight-stranded antiparallel beta-sheet flanked by seven alpha-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains.

Amino Acid Sequence↗
PubMed2001

Phenylbutazone radicals inactivate creatine kinase.

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by phenylbutazone (PB), an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H(2)O2). PB inactivated CK during its interaction with HRP-H(2) O(2), and inactivated CK in rat heart homogenate. PB carbon-centered radicals were formed during the interaction of PB with HRP-H(2)O2. The CK efficiently reduced electron spin resonance signals of the PB carbon-centered radicals. The spin trap agent 2-methyl-2-nitrosopropane strongly prevented CK inactivation. These results show that CK was inactivated through interaction with PB carbon-centered radicals. Sulfhydryl groups and tryptophan residues in CK were lost during the interaction of PB with HRP-H(2)O2, suggesting that cysteine and tryptophan residues are oxidized by PB carbon-centered radicals. Other enzymes, including alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, but not lactate dehydrogenase, were also inactivated. Sulfhydryl enzymes seem to be sensitive to attack by PB carbon-centered radicals. Inhibition of SH enzymes may explain some of the deleterious effects induced by PB.

Alcohol Dehydrogenase↗
PubMed2001

Relationship between kinetic and equilibrium folding intermediates of creatine kinase.

Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.

Creatine Kinase↗
PubMed1989

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.

Amino Acid Sequence↗
PubMed2006

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase.

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.

Cloning, Molecular↗
PubMed2003

Glycolytic buffering affects cardiac bioenergetic signaling and contractile reserve similar to creatine kinase.

Creatine kinase (CK) and glycolysis represent important energy-buffering processes in the cardiac myocyte. Although the role of compartmentalized CK in energy transfer has been investigated intensely, similar duties for intracellular glycolysis have not been demonstrated. By measuring the response time of mitochondrial oxygen consumption to dynamic workload jumps (tmito) in isolated rabbit hearts, we studied the effect of inhibiting energetic systems (CK and/or glycolysis) on transcytosolic signal transduction that couples cytosolic ATP hydrolysis to activation of oxidative phosphorylation. Tyrode-perfused hearts were exposed to 15 min of the following: 1) 0.4 mM iodoacetamide (IA; n = 6) to block CK (CK activity <3% vs. control), 2) 0.3 mM iodoacetic acid (IAA; n = 5) to inhibit glycolysis (GAPDH activity <3% vs. control), or 3) vehicle (control, n = 7) at 37 degrees C. Pretreatment tmito was similar across groups at 4.3 +/- 0.3 s (means +/- SE). No change in tmito was observed in control hearts; however, in IAA- and IA-treated hearts, tmito decreased by 15 +/- 3% and 40 +/- 5%, respectively (P < 0.05 vs. control), indicating quicker energy supply-demand signaling in the absence of ADP/ATP buffering by CK or glycolysis. The faster response times in IAA and IA groups were independent of the size of the workload jump, and the increase in myocardial oxygen consumption during workload steps was unaffected by CK or glycolysis blockade. Contractile function was compromised by IAA and IA treatment versus control, with contractile reserve (defined as increase in rate-pressure product during a standard heart rate jump) reduced to 80 +/- 8% and 80 +/- 10% of baseline, respectively (P < 0.05 vs. control), and significant elevations in end-diastolic pressure, suggesting raised ADP concentration. These results demonstrate that buffering of phosphate metabolites by glycolysis in the cytosol contributes appreciably to slower mitochondrial activation and may enhance contractile efficiency during increased cardiac workloads. Glycolysis may therefore play a role similar to CK in heart muscle.

Animals↗
PubMed2004

Evolution and divergence of the genes for cytoplasmic, mitochondrial, and flagellar creatine kinases.

Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intracellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodimers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet another gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in protochordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a protochordate tunicate. These new sequences, coupled with available sequences in the databases and sequences extracted from genome sequencing projects, provide revealing insights into the evolution and divergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved after the divergence of the craniates from the protochordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in structure consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flagellar CK genes are rather ancient and that there has been a systematic pattern of duplication and divergence consistent with changing nature of energy demands and physicochemical environment in the cells where they are expressed.

Amino Acid Sequence↗
PubMed1992

Creatine kinase and creatine kinase-MB isoenzyme during and after exercise testing in normal and obese young people.

We report creatine kinase (CK) and CK-MB values during a cycloergometric test up to maximal work capacity in 10 normal subjects aged 20 to 39 years (mean body mass index, 22 kg/m2) and 11 obese patients aged 17 to 42 years (mean body mass index, 41 kg/m2), all without any cardiorespiratory diseases. Total CK was significantly higher in obese patients. The CK-MB was not significantly different between the two groups, except at the first recovery when it was increased in obese patients and decreased in normal subjects. These results could be due to more important total stress of the total musculature, especially cardiac, and especially cardiac musculature in obese patients during a physical effort. Considering the mean values of total CK of our obese patients, it may be possible that they have myocardial damage at percentages of CK-MB less than those of lean subjects generally accepted at more than 4 percent. Moreover, in obese heart patients myocardial distress during exercise testing may be present despite heart rate at peak exercise beneath the theoretic maximal.

Adolescent↗
PubMed1996

Creatine kinase and creatine kinase-MB release after nontraumatic cardiac arrest.

The aim of the study was to describe the course of serum creatine kinase (CK) and its MB fraction (CK-MB) in patients surviving cardiac arrest, and to identify factors influencing CK and CK-MB release. The study was set in the community of Vienna, Austria. Data concerning cardiopulmonary resuscitation, collected within a period of 33 months, were evaluated retrospectively and compared with laboratory blood investigations collected prospectively (on admission and after 6, 12, and 24 hours) in 107 adult patients surviving a witnessed cardiac arrest for 24 hours. CK and CK-MB were elevated in >75% of the patients within 24 hours. Release of CK and CK-MB was mainly associated with electrocardiographic evidence of acute myocardial infarction (AMI) cumulative energy administered during defibrillation, and duration of chest trauma by compression. The CK-MB/CK ratio was elevated in 32% of the patients. Of patients with electrocardiographic evidence of AMI, only 49% had an elevated CK-MB/CK ratio. In conclusion, the elevation in serum CK and CK-MB fraction in patients after nontraumatic cardiac arrest is a frequent finding, and is associated with ischemic myocardial injury, as well as physical trauma to the chest. This should be considered when interpreting the course of CK and CK-MB fraction for the diagnosis of AMI.

Adult↗
PubMed1987

Evaluation of creatine kinase and creatine kinase-MB for diagnosing myocardial infarction. Clinical impact in the emergency room.

We prospectively studied the performance of emergency room strategies using a single sampling of total creatine kinase (CK) only and total CK with, if total CK levels were elevated, CK-MB levels in 639 patients with acute chest pain, including 386 patients who were admitted and 253 patients who were discharged. Acute myocardial infarction was diagnosed in 104 patients and excluded in 535. An elevated total CK level had a sensitivity of only 38% and specificity of only 80%, whereas a CK-MB level over 5% of an elevated total CK level had a sensitivity of only 34% and specificity of 88%. The sensitivities of both CK and CK-MB were higher in patients who arrived more than four hours after the onset of symptoms, and, in this population, the strategy using CK-MB performed significantly better than the strategy using total CK alone. Since a very positive CK-MB in a low-risk patient can greatly raise the probability of myocardial infarction, future strategies using CK-MB may have a role in selected subsets in determining which patients should not be sent home. However, the sensitivity of a single sampling of CK and CK-MB is too low for these assays to be used to exclude myocardial infarction in the emergency room or to be used as the rationale for deciding not to admit a patient.

Adult↗
PubMed1991

Simultaneous occurrence of the MB isoenzyme of creatine kinase and macro creatine kinase type 1 in a patient with acute myocardial infarction.

When the immunoinhibition method is used for differentiation of isoenzymes of creatine kinase, the simultaneous occurrence of the MB isoenzyme and variants of macro creatine kinase may lead to misinterpretation in the diagnosis of acute myocardial infarction. We describe the case of an elderly woman in whom the MB isoenzyme and the type 1 variant of macro creatine kinase were found simultaneously in her serum after acute myocardial infarction. It proved possible to identify the MB isoenzyme rapidly using a fast enzymeimmunoassay.

Aged↗
PubMed1987

Comparison of early myocardial technetium-99m pyrophosphate uptake to early peaking of creatine kinase and creatine kinase-MB as indicators of early reperfusion in acute myocardial infarction.

The value of technetium-99m pyrophosphate (Tc-99m-PYP) scintigraphy as an indicator of reperfusion 2.8 to 8 hours after the onset of symptoms of acute myocardial infarction was compared with the value of early peak creatine kinase (CK) and CK-MB release within 16 hours after the onset of symptoms. In 29 patients who received thrombolytic therapy, recanalization was seen (group 1) and in 7 it was not (group 2). In 23 patients (79%) in group 1 scintigraphic findings were positive and in all 7 in group 2 they were negative. In 15 patients (52%) in group 1 and 1 patient (14%) in group 2, CK reached its peak level within 16 hours. In 20 patients (69%) in group 1 and 3 (43%) in group 2 the CK-MB level reached a peak within 16 hours. The sensitivity, specificity and predictive accuracy of positive results of early Tc-99m-PYP scintigraphy in predicting the reperfusion were 79%, 100% and 83%. These values are significantly higher than or similar to those of early peaking of CK and CK-MB release. In contrast to measurements of enzyme release, reperfusion data for Tc-99m-PYP scintigraphy are available immediately after thrombolytic therapy. Therefore, early Tc-99m-PYP scintigraphy (3 to 8 hours after onset of symptoms) is valuable as a noninvasive technique for early diagnosis of reperfusion.

Aged↗
PubMed1998

Effect of DC shock on serum levels of total creatine kinase, MB-creatine kinase mass and troponin T.

After successful resuscitation from cardiac arrest, it is important to identify whether the event has been triggered by a myocardial infarction, since this determines subsequent investigations and management. Previous studies have shown that biochemical indices of infarction become elevated after resuscitation in patients without myocardial infarction. This can lead to overdiagnosis of myocardial infarction in the post-arrest setting. The cause of the elevated enzyme levels is not known, but may involve electrical or mechanical injury to the heart during resuscitation. In this study we aimed to identify the effects of isolated direct current shock on serum levels of creatine kinase (CK), MB creatine kinase mass (MB-CK), and troponin T, and examined the relationships between enzyme levels and the dose of electrical energy used. Thirteen patients were studied who underwent DC cardioversion for atrial fibrillation. Serum was obtained for CK, MB-CK and troponin T estimation before and 10 min after cardioversion, at hourly intervals for 8 h, and 18 h after cardioversion. Total serum CK became significantly elevated after only 3 h and rose to a peak of 1294.4 IU l(-1) (P < 0.02) at 18 h. Post-shock CK levels were strongly correlated with total shock energy (r = 0.8, P < 0.01). Serum MB-CK was significantly elevated at 18 h among patients receiving total shock energies greater than 1000 J than in those receiving lower doses, reflecting a positive correlation (r = 0.64, P < 0.05) between shock energy and peak MB-CK level. Troponin T levels were not significantly elevated after cardioversion. In conclusion, total serum CK levels become significantly elevated early after cardioversion, suggesting rapid wash-out from injured skeletal muscle. MB-CK levels become significantly elevated in individuals receiving high energy shocks, probably due to release of small quantities of the CK-MB isoform from skeletal muscle. The negligible troponin T levels seen after high energy cardioversion indicate that significant myocardial injury does not occur. Electrical injury is not likely to account for the elevated troponin T levels seen after out-of-hospital resuscitation in patients without myocardial infarction.

Aged↗